土拨鼠肝炎病毒转基因小鼠模型的建立

Establishment of Woodchuck Hepatitis Virus Transgenic Mouse Model

目的：建立土拨鼠肝炎病毒（Woodchuck hepatitis virus,WHV）转基因小鼠模型,以用于嗜肝病毒感染的发病机制研究。方法：采用微注射技术将1.3-倍WHV基因[野生型和WHV S抗原（WHV surface antigen,WHsAg）缺陷型]注射入C57BL/6xC3H小鼠的胚胎,然后与C57BL/6小鼠回交,建立WHV转基因小鼠。肝内WHV DNA复制和mRNA表达水平分别采用Southern杂交和real-time RT-PCR检测,血清中WHV病毒载量采用real-time PCR检测,HE染色进行肝组织病理学检测,ELISA检测血清中WHV核心抗体,流式细胞仪检测WHV核心抗原（WHV core antigen,WHcAg）特异性细胞毒性T淋巴细胞。结果：成功建立野生型WHV转基因小鼠和WHsAg缺陷型转基因小鼠。WHsAg缺陷型小鼠肝内WHV复制水平显著高于野生型小鼠,野生型WHV转基因小鼠血清WHV载量104-108copies/mL。而且雄性小鼠肝内和血清WHV复制水平显著高于雌性小鼠。16周龄以上的WHV转基因小鼠约半数血清可检测出高滴度的WHV核心抗体。而且少数转基因小鼠可以检测到WHcAg特异性细胞毒性T淋巴细胞。结论：这种新建的WHV转基因小鼠肝内可检测到病毒复制和mRNA表达,血清有高水平的WHV病毒载量,可以用于嗜肝病毒感染的发病机制、抗病毒治疗等研究。

Objective To establish woodchuck hepatitis virus（ WHV） transgenic（ Tg） mouse models for the study of the pathogenesis associated with hepadnaviral infection. Methods The 1. 3-fold WHV DNA constructs（ wild type WHV and mutated WHV genome lacking surface antigen） were microinjected into C57 BL /6xC3 H mice embryos to make Tg mice,which were then backcrossed with C57 BL /6. Intrahepatic WHV replication intermediates and mRNA expression were detected by Southern blotting and real-time RT-PCR,respectively. Serum WHV DNA was detected by real-time PCR. Histological examination of liver tissues was carried out with Hematoxylin-Eosin staining. Serum antibodies to WHV core antigen（ WHcAg）（ anti-WHc） were detected by ELISA. WHcAg-specific cytotoxic T lymphocytes（ CTLs） were detected by flow cytometry.Results Two lineages of WHV Tg mice harboring the WHV wild-type（ 1217） and a mutated WHV genome lacking surface antigen（ 1281） were generated. WHV replication intermediates were detected in the liver of both lineages of the Tg mice,which were much higher in 1281 than in 1217 mice. Serum WHV loads were detected at the levels of 104- 108 copies /mL in 1217 mice. Significant higher levels of intrahepatic and serum WHV DNA were detected in male Tg mice than in females.More than 50% of the mice older than 16 weeks of age showed high titers of serum anti-WHc antibodies. Moreover,WHcAgspecific CTLs could be detected in a few Tg mice. Conclusion The novel WHV Tg mouse models harbor high levels of intrahepatic WHV replication and mRNA expression,as well as serum viral loads,so that they can be used for the studies on pathogenesis related to hepadnaviral infection and antiviral therapies.